The present invention relates to fragments of a hypersensitive response elicitor which fragments elicit a hypersensitive response and uses thereof.
Interactions between bacterial pathogens and their plant hosts generally fall into two categories: (1) compatible (pathogen-host), leading to intercellular bacterial growth, symptom development, and disease development in the host plant; and (2) incompatible (pathogen-nonhost), resulting in the hypersensitive response, a particular type of incompatible interaction occurring, without progressive disease symptoms. During compatible interactions on host plants, bacterial populations increase dramatically and progressive symptoms occur. During incompatible interactions, bacterial populations do not increase, and progressive symptoms do not occur.
The hypersensitive response is a rapid, localized necrosis that is associated with the active defense of plants against many pathogens (Kiraly, Z., xe2x80x9cDefenses Triggered by the Invader: Hypersensitivity,xe2x80x9d pages 201-224 in: Plant Disease: An Advanced Treatise, Vol. 5, J. G. Horsfall and E. B. Cowling, ed. Academic Press New York (1980); Klement, Z., xe2x80x9cHypersensitivity,xe2x80x9d pages 149-177 in: Phytopathogenic Prokaryotes, Vol. 2, M. S. Mount and G. H. Lacy, ed. Academic Press, New York (1982)). The hypersensitive response elicited by bacteria is readily observed as a tissue collapse if high concentrations (xe2x89xa7107 cells/ml) of a limited host-range pathogen like Pseudonmonas syringae or Erwinia amylovora are infiltrated into the leaves of nonhost plants (necrosis occurs only in isolated plant cells at lower levels of inoculum) (Klement, Z. xe2x80x9cRapid Detection of Pathogenicity of Phytopathogenic Pseudomonads,xe2x80x9d Nature 199:299-300; Klement, et al., xe2x80x9cHypersensitive Reaction Induced by Phytopathogenic Bacteria in the Tobacco Leaf,xe2x80x9d Phytopathology 54:474-477 (1963); Turner, et al., xe2x80x9cThe Quantitative Relation Between Plant and Bacterial Cells Involved in the Hypersensitive Reaction,xe2x80x9d Phytopathology 64:885-890 (1974); Klement, Z., xe2x80x9cHypersensitivity,xe2x80x9d pages 149-177 in Phytopathoenic Prokaryotes, Vol. 2., M. S. Mount and G. H. Lacy, ed. Academic Press, New York (1982)). The capacities to elicit the hypersensitive response in a nonhost and be pathogenic in a host appear linked. As noted by Klement, Z., xe2x80x9cHypersensitivity,xe2x80x9d pages 149-177 in Phytopathogenic Prokaryotes, Vol. 2., M. S. Mount and G. H. Lacy, ed. Academic Press, New York, these pathogens also cause physiologically similar, albeit delayed, necroses in their interactions with compatible hosts. Furthermore, the ability to produce the hypersensitive response or pathogenesis is dependent on a common set of genes, denoted hrp (Lindgren, P. B., et al., xe2x80x9cGene Cluster of Pseudomonas syringae pv. xe2x80x98phaseolicolaxe2x80x99 Controls Pathogenicity of Bean Plants and Hypersensitivity on Nonhost Plants,xe2x80x9d J. Bacteriol. 168:512-22 (1986); Willis, D. K., et al., xe2x80x9chrp Genes of Phytopathogenic Bacteria,xe2x80x9d Mol. Plant-Microbe Interact. 4:132-138 (1991)). Consequently, the hypersensitive response may hold clues to both the nature of plant defense and the basis for bacterial pathogenicity.
The hrp genes are widespread in gram-negative plant pathogens, where they are clustered, conserved, and in some cases interchangeable (Willis, D. K., et al., xe2x80x9chrp Genes of Phytopathogenic Bacteria,xe2x80x9d Mol. Plant-Microbe Interact. 4:132-138 (1991); Bonas, U., xe2x80x9chrp Genes of Phytopathogenic Bacteria,xe2x80x9d pages 79-98 in: Current Topics in Microbiology and Immunology: Bacterial Pathogenesis of Plants and Animalsxe2x80x94Molecular and Cellular Mechanisms, J. L. Dangl, ed. Springer-Verlag, Berlin (1994)). Several hrp genes encode components of a protein secretion pathway similar to one used by Yersinia, Shigella, and Salmonella spp. to secrete proteins essential in animal diseases (Van Gijsegem, et al., xe2x80x9cEvolutionary Conservation of Pathogenicity Determinants Among Plant and Animal Pathogenic Bacteria,xe2x80x9d Trends Microbiol. 1:175-180 (1993)). In E. amylovora, P. syringae, and P. solanacearum, hrp genes have been shown to control the production and secretion of glycine-rich, protein elicitors of the hypersensitive response (He, S. Y., et al. xe2x80x9cPseudomonas Syringae pv. Syringae HarpinPss: a Protein that is Secreted via the Hrp Pathway and Elicits the Hypersensitive Response in Plants,xe2x80x9d Cell 73:1255-1266 (1993), Wei, Z.-H., et al., xe2x80x9cHrpI of Erwinia amylovora Functions in Secretion of Harpin and is a Member of a New Protein Family,xe2x80x9d J. Bacteriol. 175:7958-7967 (1993); Arlat, M. et al. xe2x80x9cPopA1, a Protein Which Induces a Hypersensitive-like Response on Specific Petunia Genotypes, is Secreted via the Hrp Pathway of Pseudomonas solanacearum,xe2x80x9d EMBO J. 13:543-553 (1994)).
The first of these proteins was discovered in E. amylovora Ea321, a bacterium that causes fire blight of rosaceous plants, and was designated harpin (Wei, Z.-M., et al, xe2x80x9cHarpin, Elicitor of the Hypersensitive Response Produced by the Plant Pathogen Erwinia amylovora,xe2x80x9d Science 257:85-88 (1992)). Mutations in the encoding hrpN gene revealed that harpin is required for E. amylovora to elicit a hypersensitive response in nonhost tobacco leaves and incite disease symptoms in highly susceptible pear fruit. The P. solanacearum GMI1000 PopA1 protein has similar physical properties and also elicits the hypersensitive response in leaves of tobacco, which is not a host of that strain (Arlat, et al. xe2x80x9cPopA1, a Protein Which Induces a Hypersensitive-like Response on Specific Petunia Genotypes, is Secreted via the Hrp Pathway of Pseudomnonas solanacearum,xe2x80x9d EMBO J. 13:543-53 (1994)). However, P. solanacearum popA mutants still elicit the hypersensitive response in tobacco and incite disease in tomato. Thus, the role of these glycine-rich hypersensitive response elicitors can vary widely among gram-negative plant pathogens.
Other plant pathogenic hypersensitive response elicitors have been isolated, cloned, and sequenced. These include: Erwinia chrysanthemi (Bauer, et. al., xe2x80x9cErwinia chrysanthemi HarpinEch: Soft-Rot Pathogenesis,xe2x80x9d MPMI 8(4): 484-91 (1995)); Erwinia carotovora (Cui, et. al., xe2x80x9cThe RsmAxe2x88x92 Mutants of Erwinia carotovora subsp. carotovora Strain Ecc71 Overexpress hrpNEcc and Elicit a Hypersensitive Reaction-like Response in Tobacco Leaves,xe2x80x9d MPMI 9(7): 565-73 (1966)); Erwinia stewartii (Ahmad, et. al., xe2x80x9cHarpin is not Necessary for the Pathogenicity of Erwinia stewartii on Maize,xe2x80x9d 8th Int""l. Cong. Molec. Plant-Microb. Inter. Jul. 14-19, 1996 and Ahmad, et. al., xe2x80x9cHarpin is not Necessary for the Pathogenicity of Erwinia stewartii on Maize,xe2x80x9d Ann. Mtg. Am. Phytopath. Soc. Jul. 27-31, 1996); and Pseudomonas syringae pv. syringae (WO 94/26782 to Cornell Research Foundation, Inc.).
The present invention seeks to identify fragments of hypersensitive response elicitor proteins or polypeptides, which fragments elicit a hypersensitive response, and uses of such fragments.
The present invention is directed to an isolated fragment of an Erwinia hypersensitive response elicitor protein or polypeptide where the fragment elicits a hypersensitive response in plants. Also disclosed are isolated DNA molecules which encode such fragments.
The fragments of hypersensitive response elicitors can be used to impart disease resistance to plants, to enhance plant growth, and/or to control insects. This involves applying the fragments in a non-infectious form to plants or plant seeds under conditions effective to impart disease resistance, to enhance plant growth, and/or to control insects on plants or plants grown from the plant seeds.
As an alternative to applying the fragments to plants or plant seeds in order to impart disease resistance, to enhance plant growth, and/or to control insects on plants, transgenic plants or plant seeds can be utilized. When utilizing transgenic plants, this involves providing a transgenic plant transformed with a DNA molecule encoding a fragment of a hypersensitive response elicitor protein or polypeptide which fragments elicit a hypersensitive response in plants and growing the plant under conditions effective to impart disease resistance, to enhance plant growth, and/or to control insects in the plants or plants grown from the plant seeds. Alternatively, a transgenic plant seed transformed with the DNA molecule encoding such a fragment can be provided and planted in soil. A plant is then propagated under conditions effective to impart disease resistance, to enhance plant growth, and/or to control insects on plants or plants grown from the plant seeds.